Effects of chemical ischemia on purine nucleotides,free radical generation,lipids peroxidation and intracellular calcium levels in C2C12 myotube derived from mouse myocytes

To elucidate the mechanisms of ischemia-mediated myopathy using in vitro model, changes of purine nucleotides, membrane lipid peroxidation(TBARS), intracellular calcium ([Ca2+]i)levels, generation of free radicals, and deoxyribonucleic acid (DNA) fragmentation were examined in mouse-derived C2C12 myotubes under the condition with an inhibition of glycolytic and oxidative metabolism as the ischemic condition. In purine nucleotides, intracellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations rapidly and significantly decreased after the treatment with ischemia. No remarkable differences were observed in other purine nucleotides, with the exception of inosine monophosphate (IMP) and extracellular hypoxanthine levels, both of which increased significantly during the ischemia. The lactate dehydrogenase activity in culture supernatant of C2C12 myotubes increased significantly from 2 to 4 hr after the ischemia. On the generation of free radicals, no spectrum was detected in supernatants throughout the observation period, whereas supernatant TBARS concentration increased rapidly and significantly after the ischemia. The relative intensity of [Ca2+]i significantly increased after the ischemia. On the fragmented deoxyribonucleic acid(DNA), no TUNEL positive cells was detected in C2C12 myotubes after 1 hr of the ischemia, however the positive cell percentage subsequently increased. From these results, it was suggested that the ischemic condition induced changes of membrane permeability and increase of [Ca2+]i, both of which lead to cell membrane damage, although a free radical generation was not detected. The ischemic condition also induced the release of substrate hypoxanthine for free radical generation and might initiate the apoptotic pathway in C2C12 myotubes.     

Muscle metabolic changes associated with long-term inhalation anaesthesia in the horse analysed by muscle biopsy and microdialysis techniques

During anaesthesia in the horse, muscle blood flow has been found to be reduced, possibly leading to hypoxia or ischaemia in the muscle. The aim of this study was to use the muscle biopsy and microdialysis techniques to determine whether long-term inhalation anaesthesia in laterally recumbent horses induces metabolic changes in gluteal muscle indicative of anaerobic metabolism. Muscle biopsies and plasma samples were taken from seven horses at the start and end of halothane anaesthesia. In six isoflurane-anaesthetised horses, given three pharmacological provocations (dobutamine, detomidine, acepromazine), repeated blood samples and microdialysis was performed during anaesthesia and muscle biopsies were taken before and at the end of anaesthesia. Adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate, inosine monophosphate (IMP) creatine phosphate and lactate concentrations did not differ between dependent and non-dependent muscles at either sampling time. Creatine phosphate decreased in both the halothane (-38%) and isoflurane (-28%) group. In the halothane group, ATP was decreased (-15%) at the end of anaesthesia, while IMP was increased (+32%). Lactate in muscle and plasma increased in both groups. Lactate in dialysate increased after induction and remained elevated above plasma concentrations. These results show that long-term inhalation anaesthesia in horses is associated with an anaerobic metabolic response within the muscle and that microdialysis can be used to detect metabolic changes within the muscle during equine anaesthesia.     

牦牛尿嘌呤衍生物排出量对皱胃注射酵母RNA水平的响应

本研究对3头牦牛施以皱胃瘘管手术,以酵母RNA为嘌呤碱基供体,连续注射4期,以期测定牦牛吸收嘌呤的回收率,为尿嘌呤衍生物估测牦牛瘤胃微生物氮产量的模型积累数据。 结果表明,牦牛皱胃连续注射酵母RNA可使尿囊素(564～1 426 μmol/kg BW^0.75)、总嘌呤衍生物(purine derivative, PD)排出量(629～1 507 μmol/kg BW^0.75)及尿囊素占总PD比例线性提高(0.90～0.95)(P<0.01);嘌呤碱基注射水平对尿酸、肌酐及尿氮排出量影响不显著(P>0.05)。回归分析发现,牦牛皱胃嘌呤注射量(X,mmol/d)与尿嘌呤衍生物排出量(Y,mmol/d) 间存在线性关系:Y=0.85X+33.02 (R²=0.96,P<0.001),牦牛吸收嘌呤在尿中的回收率为85%。     

Effects of submaximal exercise on adenine nucleotide concentrations in skeletal muscle fibers of horses with polysaccharide storage myopathy

OBJECTIVE: To determine whether disruption of adenine triphosphate (ATP) regeneration and subsequent adenine nucleotide degradation are potential mechanisms for rhabdomyolysis in horses with polysaccharide storage myopathy (PSSM) performing submaximal exercise. ANIMALS: 7 horses with PSSM and 4 control horses. PROCEDURES: Horses with PSSM performed 2-minute intervals of a walk and trot exercise on a treadmill until muscle cramping developed. Control horses exercised similarly for 20 minutes. Serum creatine kinase (CK) activity was measured 4 hours after exercise. Citrate synthase (CS), 3-OH-acylCoA dehydrogenase, and lactate dehydrogenase activities prior to exercise and glucose-6-phosphate (G-6-P) and lactate concentrations before and after exercise were measured in gluteal muscle specimens. Adenine triphosphate, diphosphate (ADP), monophosphate (AMP), and inosine monophosphate (IMP) concentrations were measured before and after exercise in whole muscle, single muscle fibers, and pooled single muscle fibers. RESULTS: Serum CK activity ranged from 255 to 22,265 U/L in horses with PSSM and 133 to 278 U/L in control horses. Muscle CS activity was lower in horses with PSSM, compared with control horses. Muscle G-6-P lactate, ATP, ADP, and AMP concentrations in whole muscle did not change with exercise in any horses. Concentration of IMP increased with exercise in whole muscle, pooled muscle fibers, and single muscle fibers in horses with PSSM. Large variations in ATP and IMP concentrations were observed within single muscle fibers. CONCLUSIONS AND CLINICAL RELEVANCE: Increased IMP concentration without depletion of ATP in individual muscle fibers of horses with PSSM during submaximal exercise indicates an energy imbalance that may contribute to the development of exercise intolerance and rhabdomyolysis.     

Effect of creatine supplementation on muscle metabolic response to a maximal treadmill exercise test in Standardbred horses

The aim of the present study was to investigate the effect of creatine (Cr) supplementation on muscle metabolic response in connection with a maximal treadmill exercise test, known to cause a marked anaerobic metabolic response and adenine nucleotide degradation. First, 6 Standardbred trotters performed a standardised maximal exercise test until fatigue (baseline test). The test used was an inclined incremental treadmill test in which the speed was increased by 1 m/s, starting at 7 m/s, every 60 s until the horse could no longer keep pace with the treadmill. After this baseline test, the horses were separated into 2 equal groups. One half received a dose of 25 g creatine monohydrate twice daily, and the other group were given the same dose of lactose (placebo). The supplementation period was 6.5 days, after which the maximal treadmill exercise test was performed again. A washout period of 14 days was allowed before treatments were switched between groups and a new supplementation period started. After this second supplementation period a new maximal exercise test was performed. After supplementation with creatine or placebo, horses were stopped after performing the same number of speed steps and duration of exercise as they had in the baseline test. Blood samples for analysis of plasma lactate, creatine (Cr), creatinine, hypoxanthine, xanthine and uric acid concentrations were collected at rest, during each speed step and during recovery. The total blood volume (TBV) was also determined. Muscle biopsies for analysis of muscle metabolites (adenosine triphosphate [ATP], adenosine diphosphate [ADP], adenosine monophosphate [AMP], inosine monophosphate [IMP], creatine phosphate [CP], lactate [La] and glycogen) were taken at rest, immediately post exercise and after 15 min recovery. The results showed no significant increase in plasma Cr or muscle total creatine concentration (TCr) after supplementation with Cr. At the end of exercise ATP and CP concentrations had decreased and IMP and lactate concentrations increased in muscle in all groups. Plasma lactate concentration increased during exercise and recovery and plasma uric acid concentration increased during recovery in all groups. No influence could be found in TBV after supplementation with creatine. These results show that creatine supplementation in the dosage used in this study had no influence on muscle metabolic response or TBV.     

Release of dopamine and ATP from PC12 cells treated with dexamethasone, reserpine and bafilomycin A1

The amounts and time courses of dopamine and ATP released from perfused PC12 cells were examined using a simultaneous on-line recording system. High KCl (60 mM) caused dopamine and ATP release with similar time courses. The relative amount of dopamine to ATP in the effluent was 9.5. In PC12 cells cultured with dexamethasone, reserpine or bafilomycin A1 for 2 days, these drugs did not affect increases of intracellular Ca2+ in response to high KCl. Dexamethasone doubled the amount of dopamine release induced by high KCl without changing the amount of ATP release. High KCl failed to cause dopamine release in reserpine-treated cells but evoked ATP release. Bafilomycin A1 decreased both high KCl-induced dopamine and ATP release. The ratio of released ATP to total adenine nucleotides and adenosine in response to high KCl was not changed by treatment with the drugs. These results suggest that dopamine and ATP are simultaneously released from secretory vesicles of PC12 cells, in which they are stored via different pathways. Similar to dopamine uptake into secretory vesicles, the H+-gradient across the vesicular membrane developed by vacuolar ATPase may play an important role in the vesicular uptake of ATP.     

Thermal stimulation at 39°C facilitates the fusion and elongation of C2C12 myoblasts

The aim of this study was to determine the effect of thermal stimulation at 39°C on the fusion and elongation of skeletal muscle cells. During a 5 day differentiation process of C2C12 cells, nine groups subjected to varying lengths of thermal stimulation at 39°C were established. Afterward, all groups were immunostained using anti‐muscle heavy‐chain antibody to test for myotube formation. Quantification of the myotube area demonstrated a significant increase in the group subjected to thermal stimulation at 39°C during the latter half of the differentiation compared with the control group, but the fusion index was significantly higher in the group that received hyperthermic treatment during the first half of the differentiation period. Moreover, the longitudinal length of myotubes was significantly increased in the groups that were subjected to thermal stimulation at 39°C during the latter half of the differentiation period. The distance between the center of myotubes and the nucleus farthest away from the center was substantially extended in the group receiving thermal stimulation at 39°C only on the fourth day of the differentiation. Together, these results demonstrate that thermal stimulation at 39°C facilitates myoblast fusion and elongation.     

Response of C2C12 mouse and turkey skeletal muscle cells to the beta-adrenergic agonist ractopamine

The effects of ractopamine (RAC) and ractopamine stereoisomers (RR, RS, SR, and SS) on cyclic AMP (cAMP) production, total protein, and DNA concentrations in mouse skeletal muscle cells (C2C12) were evaluated. The RAC (10 microM) caused an approximately 30% increase in cell number, protein, and DNA concentrations in myoblasts after 48 h; no differences were found in myotubes. The RAC-stimulated increase of these variables in myoblasts was blocked by the presence of equimolar concentrations of propranolol. At a later passage, myoblasts failed to exhibit an increase in cell number, protein, or DNA upon exposure to RAC. Both myoblasts and myotubes increased cAMP production in response to 10 microM RAC. The RAC isomers ranked RR > SR > RS approximately SS in ability to stimulate cAMP production, with essentially no response to SS. The SR produced about 50% of the RR response. Coincubation of propranolol (10 microM) and RAC (10 microM) prevented RAC-stimulated cAMP production in myotubes but not in myoblasts (approximately 35% of cAMP produced by RAC alone). Turkey satellite cells (derived from biceps femoris of 12-wk-old toms) produced essentially no increased cAMP when exposed to 10 microM RAC stereoisomers. Stability of RAC was evaluated under laboratory storage and culture conditions. The RAC was stable for more than 4 mo when stored in deuterated DMSO (>98% purity) at room temperature or in aqueous solutions at -80 degrees C, as determined from sequential nuclear magnetic resonance studies. Radiolabeled RAC was incubated for 72 h in the presence of serum-containing medium, with or without C2C12 cells. Ninety-eight percent of the parent compound found in the medium at time zero was present in the medium as parent at the end of 72 h. The cellular cAMP response to RAC through beta-adrenergic receptors seems to be stereospecific. If the state of myoblasts and myotubes in vitro reflects the in vivo state, then the ractopamine effect in vivo on cellular processes (including cell division and protein and DNA accumulation) may be independent of beta-adrenergic receptors in muscle.     

Urinary purine derivates excretion as an indicator of in vivo microbial N flow in cattle: A review

Microbial protein flow to the duodenum may be regarded as the most important and sensitive indicator to optimise rumen metabolism in high-yielding dairy cows. In this review, the methodology and the sources of variation to estimate the duodenal microbial N flow with urinary excretion of purine derivatives (PD) as a non-invasive method is discussed. The urinary PD excretion was linearly related with the amount of purine bases (PB) infused in the abomasum or duodenum, but the recovery of PB in urine differed between experiments. The main sources of variation in the relationship between microbial N flow and urinary PD excretion are dietary contribution of nucleic acids to duodenal flow, varying N:purine ratio in duodenal digesta, differences in intestinal digestibility of nucleic acids and infused PB, and endogenous contribution of PD to urinary excretion. The recycling of PD to the rumen is negligible, and does not explain the incomplete urinary recovery of PD. A large proportion of the total PD is excreted as allantoin in urine. In some experiments this proportion was constant, whereas in others it varied with diet or physiological state of the animal. The excretion of PD in milk is not a suitable indicator of microbial N flow, due to mammary purine catabolism to uric acid and due to the strong positive correlation between milk allantoin excretion and milk yield. Instead of total urine collection, the molar ratio between urinary PD and creatinine can be used to estimate microbial N flow. However, a substantial between-animal variation in this ratio was found, and effects of changes in energy balance of dairy cows on urinary creatinine excretion should be determined. The urinary excretion of total PD and of allantoin provided lower estimates of duodenal microbial N flow than with measurements in the omasum or duodenum, but they closely reflected the changes observed with these measurements.     

Urinary excretion of purine derivatives and plasma allantoin level in sheep and goats during fasting

The relationship between blood plasma level and urinary excretion of allantoin (AN) was examined in sheep and goats during fasting to investigate the possible use of purine derivatives (PD) in urine and/or plasma for estimating the microbial protein production in the rumen, and the further digestion in the lower guts of ruminants. Urinary AN excretion decreased markedly during fasting (0.13 mmol/kgW^0.75 per day), although urinary levels of other PD, hypoxanthine + xanthine and uric acid did not differ irrespective of the feeding condition, that is, feeding, fasting and refeeding in both species. The AN concentration in blood plasma also decreased drastically in the starvation period, and was suddenly increased on refeeding in sheep and goats, and these phenomena were very similar to those of urinary AN excretion. Therefore, there was a high positive correlation between plasma AN level and urinary AN excretion, and the coefficient of correlation was statistically significant (P < 0.01). These results clearly indicate that changes in urinary AN reflect change in plasma AN, which is induced by the catabolism of purine base in the body.     

The effect of exogenous purine supply on the endogenous excretion of purine derivatives in the urine of growing lambs

An experiment was made to determine the absorption of purine metabolites in dietary nucleic acids through the digestive tract, and also to determine the utilization of nucleic acids absorbed in the body, using growing lambs. Two pairs of 120‐ and 180‐day‐old twin female lambs with a bodyweight of 18.2–19.0 kg were kept in metabolism crates and fed on purine‐free milk replacement (MR) with supplements of exogenous purine (purine base or purine nucleoside) at a level of 0.2 mmol/BW^0.75/d for 5 consecutive days, and thereafter they were maintained in the crates for 4 days. The daily amount of exogenous purine supply was calculated based on the urinary excreted purine derivatives (PD) in lambs fed on milk replacement alone. A urine sample was collected daily for 9 consecutive days, and the urinary excretion of PD was determined daily. Urinary PD excretion opened to increase within 24 h after the dose of purine bases, and the level was recovered on 3 days after ceasing the exogenous purine supply. The recovery of PD in the urine was about 70% of the purine supplement. When purine nucleosides were added to the feed, urinary PD excretion was initiated within 24 h after dosing, and the values were recovered after ending the purine nucleoside supply. The recovery rate of PD in the urine was only 30% of the supplemented purine. The plasma allantoin levels were almost similar after feeding purine bases and purine nucleosides, and the values were mostly in the range (40–60 µmol/L). These findings indicate that an exogenous purine can be directly incorporated into the body, and the purine as nucleoside is more effectively utilized for the synthesis of nucleic acids than as a purine base in the body of growing lambs.     

Cardiovascular Responses to Glibenclamide During Endotoxaemia in the Pig

Vanelli, G., Hussain, S.N.A., Dimori, M. and Aguggini, G., 1996. Cardiovascular responses to glibenclamide during endotoxaemia in the pig. Veterinary Research Communications, 21(3), 187-200The effects of blockading the ATP-sensitive potassium channel (K+ATP channels) on endotoxin-induced vascular derangements was studied. Escherichia coli endotoxin was infused (20 µg/kg per h) intravenously for 180 min into anaesthetized, mechanically ventilated, indomethacin-treated pigs. After 150 min of endotoxaemia, glibenclamide (a K+ATP channel blocker) was infused intravenously at 2 mg/kg per min for 5 min. The cardiovascular parameters were recorded before (control), every 30 min up to 150 min during endotoxaemia, and then at 5, 15 and 30 min after administration of glibenclamide. Infusion of endotoxin reduced the systemic arterial pressure to 60.6% ± 3.7% (p < 0.01) and increased the pulmonary arterial pressure by 75.9% ± 11.0% (p < 0.01) of the control values. Within 5 min, infusion of glibenclamide transiently but significantly reversed the systemic hypotension by raising the systemic vascular resistance, whereas the increased pulmonary arterial pressure was further augmented. Glibenclamide infusion did not influence the cardiac output. Within 30 min, the cardiovascular parameters had returned to the values induced by endotoxin, except for the systemic vascular resistance. Infusion of glibenclamide into normal pigs did not change the systemic pressure or resistance, but the pulmonary pressure and resistance were augmented transiently. These data suggest that, in pigs, the ATP-sensitive K+ channels may be one factor playing a role in the vascular changes due to endotoxaemia, especially in the systemic circulation.     

Development of methods to measure activity of polysomes and cytoplasmic enzymes from bovine skeletal muscle in in vitro protein-synthesis assays

A cell-free, protein-synthesis system containing components from bovine skeletal muscle was developed as prerequisite to attempts to learn whether polysomes or cytoplasmic enzymes limit rate of muscle protein synthesis in the current population of domestic animals. Amino acid incorporation into trichloroacetic acid-precipitable protein was optimal at pH 7.5 and, if Tris buffer was used, at K+ and Mg2+ concentrations of 40 mM and 4 mM, respectively. Optimal concentrations of compounds that provide energy for amino acid incorporation were 1 mM ATP, .2 mM GTP, 20 micrograms creatine phosphokinase/ml and 20 mM creatine phosphate; .03 mM of each of the 20 amino acids was required for assays lasting up to 60 min. Neither rate of tRNA acylation nor availability of aminoacyl-tRNA was rate-limiting in the cell-free system established; hence, the cytoplasmic enzyme fraction from bovine skeletal muscle contains ample tRNA and has aminoacyl-tRNA synthases that are sufficiently active to form aminoacyl-tRNA faster than these compounds are used to form polypeptides in the cell-free system developed. Reinitiation of ribosomes onto new mRNA occurred very slowly, if at all, in the protein-synthesis system developed. Cytoplasmic enzymes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 3.2:1 or lower in the cell-free system. Polysomes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 400:1 or higher in the cell-free system. These ratios define the conditions needed to assay activity of cytoplasmic enzymes or polysomes from different animals quantitatively.     

快速估测反刍动物瘤胃微生物蛋白在小肠流量的方法

选用瘤胃尚未发育的初生小尾寒羊羔羊及梅花仔鹿各4只,人工哺乳,随牛奶分别饲喂给羔羊和仔鹿5个水平的核酸。羔羊尿中嘌呤衍生物的总量(Y,mmol/d)随着摄入嘌呤量(X,mmol/d)的增加而增加。其线性方程为:Y=0.752+0.662X(r=0.995,P<0.001)。仔鹿尿中嘌呤衍生物的总量(Y,mmol/d)随着摄入嘌呤量(X,mmol/d)的增加也明显增加,线性方程为:Y=0.559+0.326X(r=0.990,P=0.001)。利用紫外分光光度计测定的羔羊尿中尿酸当量值与嘌呤摄入量呈强正相关(r=0.994,P<0.01)。由此推算出利用尿中尿酸当量值来估测羊小肠微生物氮流量(Y,g/d)的方程为:Y=0.417+0.396X;推算出的利用尿中尿酸当量值估测鹿小肠微生物氮流量(Y,g/d)的方程为:Y=-4.137+1.245X。X(mmol/d)为测得的羊或鹿尿中尿酸当量值。     

Porcine leptin inhibits protein breakdown and stimulates fatty acid oxidation in C2C12 myotubes

This study evaluated the potential mechanism(s) by which leptin treatment inhibits loss of muscle mass with fasting. Cultures of C2C12 myoblasts were differentiated into myotubes with 5% (vol/vol) horse serum in Dulbecco's modified Eagle's medium/F12. These myotubes were used to assess 3H-tyrosine incorporation and release following incubation with recombinant porcine leptin (0 to 500 ng/mL). Protein synthesis in myotubes, as measured by 3H-tyrosine incorporation, was not affected by leptin treatment (P > 0.05). Protein breakdown in C2C12 myotubes, as measured by 3H-tyrosine release, was inhibited by leptin treatment. A leptin concentration of 0.5 ng/mL was sufficient to inhibit 3H-tyrosine release by 3.5% (P < 0.05); 50 ng/mL produced a maximal inhibition of 10.2% (P < 0.05). Dexamethasone (1 microM) was used to maximally stimulate protein breakdown. Leptin (50 ng/mL leptin) decreased dexamethasone-induced 3H-tyrosine release by 32% (P < 0.05). The inhibition of 3H-tyrosine release in C2C12 myotubes suggests that leptin produces a protein-sparing effect in vitro by inhibiting protein breakdown. Fatty acid metabolism also was investigated because fatty acids are a major energy source for muscle during periods of reduced intake, as occurs with leptin treatment. Acute (4 h) and chronic (24 h) exposures to porcine leptin (0 to 500 ng/mL) were used to evaluate 14C-palmitate oxidation. Acute leptin treatment had no effect (P > 0.05) on palmitate metabolism. Chronic leptin exposure resulted in up to a 26% increase in palmitate oxidation (P < 0.05). The stimulation of fatty acid oxidation with chronic leptin treatment suggests that leptin spares other energy sources in muscle from oxidation during periods of a leptin-induced decrease in feed intake.     

Myogenic and adipogenic properties of goat skeletal muscle stem cells

The aims of the present study were to establish a culture system for goat skeletal muscle stem cells and to examine their myogenic and adipogenic properties in vitro. Cells were isolated from the skeletal muscle of the Shiba goat and cultured in vitro. Most of the cells were positive for myogenic markers, such as Pax7, MyoD, and desmin, and immunocytochemistry revealed they differentiated to form myotubes expressing myosin heavy chain, indicating they were highly myogenic. Myogenic differentiation was strongly suppressed by the addition of basic fibroblast growth factor, while proliferation was unaffected. When the cells were cultured in adipogenic differentiation medium, some of the cells differentiated into mature adipocytes that stained with Oil Red-O. These cells were immunocytochemically positive for adipogenic markers, including peroxisome proliferator-activated receptor-gamma (PPAR gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP alpha). These results clearly demonstrate the presence of both myogenic and adipogenic stem cells in goat skeletal muscle.     

Pig blastocyst development in vitro is affected by amino acids

Pig embryos were removed 5 d after onset of estrus and cultured in modified Krebs-Ringer bicarbonate (mKRB) medium containing bovine serum albumin (BSA; 1 mg/ml). Media in Exp. 1 and 2, but not in Exp. 3, contained 10% heat-inactivated lamb serum (LS). In Exp. 1, embryos were cultured in 1) mKRB + LS or mKRB + LS supplemented with 2) glutamine (2 mM); 3) phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); or 4) phenylalanine, methionine, isoleucine and glutamine. Embryos cultured in media with supplemental phenylalanine, methionine and isoleucine attained larger (P less than .05) volumes during culture and initiated hatching at an increased (P less than .05) frequency compared with embryos cultured without additional amino acids. In contrast, adding glutamine depressed (P less than .05) the maximum volumes observed during culture. In Exp. 2 mKRB + LS was compared with mKRB + LS plus phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM); methionine and isoleucine; phenylalanine and isoleucine, or phenylalanine and methionine. Embryos cultured in media supplemented with phenylalanine and methionine attained larger (P less than .05) volumes than embryos cultured in either media without added methionine. Fewer (P less than .05) embryos cultured without methionine initiated hatching (56%) compared with embryos provided methionine (89%). In Exp. 3, we evaluated the addition of glutamine (1 mM) with or without phenylalanine (.1 mM), methionine (.05 mM) and isoleucine (.2 mM) to serum-free mKRB. Adding glutamine alone, but not in combination, increased (P less than .05) blastocyst volumes on d 3 and maximum volume attained during culture compared with embryos cultured in mKRB alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum Metabolomics Analysis of Feline Mammary Carcinomas Based on LC-MS Techniques

The purpose of this study was to investigate the variation of differential metabolites in mammary carcinomas bearing cats and healthy cats, and to explore the relationship between differential metabolites and the occurrence of feline mammary carcinomas. In this study, 6 feline mammary carcinomas serum samples were selected as test (T) group. Meanwhile, 6 healthy cats with similar age and same breed were selected as control (C) group. Serum samples were detected by ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (LC-MS). Differential metabolites were screened by principal component analysis (PCA), orthogonal projections to latent structures-discriminant analysis (OPLS-DA) and Student's t-test. Then the hierarchical clustering analysis (HCA) was carried out for the screened differential metabolites. The KEGG annotation and the metabolic pathway of differential metabolites were analyzed. The results showed that a total of 159 differential metabolites were identified in the 2 groups. Compared with C group, 49 differential metabolites were down-regulated and 110 differential metabolites were up-regulated in T group. Finally, a total of 5 differential metabolites which closely related to feline mammary carcinomas were selected. Ergothioneine (EGT) and creatine in T group were down-regulated, while indolelactic acid (IAA), choline and uric acid were up-regulated compared to C group. These differential metabolites indicated that during the development of feline mammary carcinomas, the body changes involved multiple metabolic pathways, such as glycine, serine and threonine metabolism, arginine and proline metabolism, histidine metabolism, tryptophan metabolism, purine metabolism and glycerophospholipid metabolism. This study provides a new idea for further research of the pathogenesis of feline mammary carcinomas.     

基于LC-MS技术的患乳腺癌猫血清代谢组学分析

本试验旨在研究患乳腺癌猫与健康猫体内差异代谢物的变化情况,并探讨差异代谢物与猫乳腺癌发生之间的关系。本试验选取临床收集的经组织病理学确诊的猫乳腺癌血清样本6例作为试验组（T组）,同时选取年龄相似,品种相同的健康猫血清6例作为对照组（C组）。运用超高效液相色谱质谱联用技术（liquid chromatography-mass spectrometry,LC-MS）对两组猫的血清样本进行检测,采用无监督的主成分分析（principal component analysis,PCA）、正交偏最小二乘法-判别分析（orthogonal projections to latent structures-discriminant analysis,OPLS-DA）及学生t检验（Student's t-test）筛选出差异代谢物,然后再对筛选出的差异代谢物进行层次聚类分析（hierarchical clustering analysis,HCA）,并进行差异代谢物的KEGG注释及差异代谢物的代谢通路分析。结果表明,在两组样本中共定性到159种差异代谢物。与C组相比,在T组中有49种差异代谢物出现下调,110种差异代谢物出现上调。最终共筛选出5种与猫乳腺癌发生发展密切相关的差异代谢产物。与C组相比,T组中麦角硫因（ergothioneine,EGT）和肌酸（creatine）出现下调,吲哚乙酸（indolelactic acid,IAA）、胆碱（choline）和尿酸（uric acid）出现上调。这些差异代谢物表明,在猫乳腺癌的发生过程中,机体变化涉及了甘氨酸、丝氨酸和苏氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、色氨酸代谢、嘌呤代谢异常和甘油磷脂代谢等多个代谢途径,为今后深入研究猫乳腺癌的发病机制开辟了一个新的思路。     

Comparisons of four methods for quantification of lysosomal cysteine proteinase activities

Four methods were compared to optimize the measurement of the activities of cathepsin B and cathepsin L in porcine skeletal muscle. These methods were: Method A (lysosomal enriched fraction obtained by differential centrifugation), Method B (muscle extract in the absence of detergent), Method C (muscle extract in the presence of detergent) and Method D (the same as method C, but passed through a S-carboxymethylated-papain-Sepharose affinity column). Results indicated that, of the methods tested, Method D yielded greater cathepsin B and consistently greater cathepsin B + L activities per gram of muscle. Hence, Method D is the method of choice for quantification of these enzyme activities. Studies indicated that for cathepsin B with Z-Arg-Arg-NMec as substrate, Km and Vmax values were .416 mM and 4,405 pmol.min-1.mg protein-1, respectively. The Km and Vmax for cathepsins B + L were .132 mM and 9,346 pmol.min-1.mg protein-1, respectively. The relationship between enzyme activity and incubation time was linear for the incubation times studied (up to 60 min). Also, the relationship between enzyme activities and amount of protein in the assay was linear at the concentrations studied (up to 20 micrograms protein). The same preparations were assayed by conditions commonly used by many investigators (.005 mM substrate, approximately 75 micrograms protein, 30 min at 37 degrees C) and by conditions established in this study (1.0 mM substrate, 10 micrograms protein and 15 min at 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)